HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Severe impairment of leukocyte rolling in venules of core 2
glucosaminyltransferase–deficient mice
Markus Sperandio, Aravinda Thatte, Dan Foy, Lesley G. Ellies, Jamey D. Marth, and Klaus Ley
Leukocyte capture and rolling are mediated by selectins expressed on leukocytes (L-selectin) and the vascular endothelium (P- and E-selectin). To investigate
the role of core 2 ␤1-6-N-glucosaminyltransferase (C2GlcNAcT-I) for synthesis
of functional selectin ligands in vivo, leukocyte rolling flux and velocity were studied in venules of untreated and tumor
necrosis factor-␣ (TNF␣)–pretreated autoperfused cremaster muscles of
C2GlcNAcT-I–deficient (core 2ⴚ/ⴚ) and littermate control mice. In untreated core
2ⴚ/ⴚ mice, leukocyte rolling was dramatically reduced with markedly increased
rolling velocities (81 ⴞ 4 ␮m/s vs 44 ⴞ 3
␮m/s). The reduced rolling in core 2ⴚ/ⴚ
mice was due mainly to severely impaired
binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1). Some rolling
remained after blocking PSGL-1 in controls but not in core 2ⴚ/ⴚ mice. In TNF␣pretreated mice, rolling was markedly reduced in core 2ⴚ/ⴚ mice owing to impaired
P-selectin– and E-selectin–mediated rolling. Rolling velocities in core 2ⴚ/ⴚ mice
treated with an E-selectin–blocking monoclonal antibody (59 ⴞ 4 ␮m/s) were significantly higher than in controls (14 ⴞ 1
␮m/s), which provides further evidence
for the severe impairment in P-selectin–
mediated rolling. In conclusion, P-selectin ligands including PSGL-1 are largely
C2GlcNAcT-I dependent. In addition, Eselectin–mediated rolling in vivo is partially dependent on the targeted
C2GlcNAcT-I. (Blood. 2001;97:3812-3819)
© 2001 by The American Society of Hematology
Introduction
Leukocyte recruitment to sites of inflammation requires a multistep
adhesion cascade beginning with leukocyte capture and rolling
leading to firm adhesion and transmigration.1,2 Capture and rolling
are mediated largely by the selectin family of adhesion molecules.
Intravital microscopy studies on inflamed tissue of the cremaster
muscle conducted in gene-targeted mice deficient in either E-, P-,
or L-selectin or various combinations have revealed both overlapping and unique functions of the selectins.3,4 The overlapping
function of selectins is in particular evident in E-selectin–deficient
mice. These mice do not show obvious inflammatory deficits in
certain models unless P-selectin is also blocked,5 which then leads
to a dramatic reduction in the number of rolling leukocytes.6
Characteristic properties of individual selectins become evident in
their ability to mediate leukocyte rolling at distinct velocities.
Untreated wild-type mice show P-selectin–dependent rolling with
typical rolling velocities in venules of the cremaster muscle of
below 50 ␮m/s.7 In tumor necrosis factor-␣ (TNF␣)–treated mice,
E-selectin preferentially mediates slow rolling (10 ␮m/s or less),6
and P-selectin mediates rolling at higher velocities.3
Selectin ligands carry sialylated and fucosylated oligosaccharides found at the nonreducing termini of core 2-dependent
O-linked glycans.8-10 Core 2 structures are formed by a family of
core 2 ␤1-6-N-glucosaminyltransferase branching enzymes that
play a key role in controlling O-glycan structural diversity.11 The
major ligand for P-selectin, P-selectin glycoprotein ligand-1 (PSGL1), is a glycoprotein with at least one sulfated tyrosine near the
N-terminus and with sialylated and fucosylated O-glycans.12,13
Previous studies on Chinese hamster ovary (CHO) cells transfected
with different fucosyltransferases and PSGL-1 have shown that
transfection of these cells with C2GlcNAcT-I, a core 2 ␤1-6-Nglucosaminyltransferase, leads to a dramatic increase in binding to
P-selectin.14 These and other findings demonstrate that PSGL-1
requires C2GlcNAcT-I for optimal binding to P-selectin.15 Recent
work by Ellies et al16 provides evidence that selectin ligand function is
impaired but not absent in neutrophils from mice deficient in
C2GlcNAcT-I. These conclusions are based on results from binding
of soluble recombinant selectins to neutrophils and from parallelplate flow chamber–controlled detachment assays showing impaired leukocyte adhesion on all 3 selectins. Leukocyte recruitment
in a thioglycollate-induced peritonitis model showed an 80% reduction
in polymorphonuclear cells, which is comparable to the defects seen in
E/P-selectin–deficient or fucosyltransferase VII–deficient mice.17-19
The present study was designed to test whether P- and/or
E-selectin ligand functions are impaired under physiological conditions in vivo by the absence of C2GlcNAcT-I. To this end, we
investigated leukocyte rolling in vivo in C2GlcNAcT-I–deficient
mice in 2 models of inflammation (untreated and TNF␣-treated
mice). Mild trauma caused by the exteriorization of the cremaster
muscle leads to a rapid induction of P-selectin–dependent leukocyte rolling7,20; E-selectin expression is absent or very low in this
model. Short-term (2-hour) TNF␣ treatment leads to the expression
of P- and E-selectin on the surface of venular endothelium,21 which
coincides with the induction of slow leukocyte rolling in these
microvessels.6
From the Department of Biomedical Engineering, University of Virginia,
Charlottesville, and the Howard Hughes Medical Institute, Department of
Cellular and Molecular Medicine, University of California, La Jolla.
Reprints: Klaus Ley, Department of Biomedical Engineering and Cardiovascular Research Center, University of Virginia, Health Sciences Center, Box
800759, Charlottesville, VA 22908; e-mail: [email protected].
Submitted November 14, 2000; accepted February 13, 2001.
Supported by National Institutes of Health (NIH) grants HL-64381 and
HL-54136 (K.L.); by NIH grant DK 48247 (J.D.M.); by a stipend from the
German Research Foundation (DFG) SP 621/1–1 (M.S.); and by National
Cancer Institute Fellowship F32CA79130 (L.G.E.).
3812
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
Materials and methods
Animals
Mice lacking a functional gene encoding core 2 ␤1-6-N-glucosaminyltransferase (C2GlcNAcT-I, EC 2.4.1.102) were derived from a colony
described previously.16 C2GlcNAcT-I alleles were analyzed by Southern
blotting and polymerase chain reaction (PCR) as described earlier.16 The
wild-type C2GlcNAcT-I allele was detected by means of PCR primers
located adjacent to the deleted region (W5⬘: 5⬘-GGGTTACGGATGAGCTCTGTGTC; and W3⬘: 5⬘-CCCTGGAAGCAGGACAATTCTG3⬘), resulting in a 304–base pair (bp) fragment, and the mutant allele was
detected by means of W5⬘ and a loxP primer (M3⬘: 5⬘-CTCGAATTGATCCCCGGGTAC-3⬘), yielding a 200-bp fragment. Control experiments were
performed in heterozygous (⫹/⫺) and homozygous (⫹/⫹) littermates. All
experiments were performed on healthy mice that were at least 8 weeks of
age. Mice were housed in a barrier facility under specific-pathogen–free
conditions, and animal experiments were approved by the animal care and
use committee of the University of Virginia.
Antibodies and cytokines
The rat antimouse E-selectin monoclonal antibody (mAb) 9A9 (rat
immunoglobulin G1 [IgG1], 30 ␮g per mouse) has previously been shown
to specifically block E-selectin function in vitro22 and E-selectin–dependent
rolling in vivo.6 The mAb 9A9 was a gift from Dr B. Wolitzky (HoffmanLa Roche, Nutley, NJ). The P-selectin mAb RB40.34 (rat IgG1, 30 ␮g per
mouse) blocks P-selectin–dependent adhesion in vitro,23 P-selectin–
dependent leukocyte rolling in vivo,7 and P-selectin–dependent leukocyte
recruitment in vivo.23 The rat antimouse mAb to PSGL-1, 4RA10 (rat IgG1,
30 ␮g per mouse), has been shown to specifically block PSGL-1 in vitro and
in vivo.24 The mAbs RB40.34 and 4RA10 were gifts from Dr Dietmar
Vestweber at the University of Münster (Germany). The L-selectin mAb
MEL14 (rat IgG2a; 50 ␮g per mouse) was purified from hybridoma
supernatant (American Type Culture Collection, Manassas, VA) and blocks
L-selectin–dependent leukocyte rolling in vivo.6,7 Recombinant murine
TNF␣ (Genzyme, Cambridge, MA) was injected intrascrotally at a dose of
500 ng per mouse in a volume of 0.3 mL sterile saline 2 hours before the
beginning of the intravital microscopic experiment.
Flow cytometry
Flow cytometry was used to find the saturating dose of PSGL-1–blocking
mAb 4RA10 after systemic injection into mice. Four anesthetized control
mice were treated with increasing doses of mAb 4RA10 (1 ␮g, 3 ␮g, 10 ␮g,
and 100 ␮g per mouse) or a rat antimouse IgG control antibody (100 ␮g)
(Pharmingen, San Diego, CA). At 20 minutes after injection of the
PSGL-1–blocking mAb 4RA10 or the isotype control, peripheral blood was
collected from the carotid artery. Red blood cells were lysed in Pharm-Lyse10X solution (Pharmingen). After centrifugation and removal of the
supernatant, blood cells (1 ⫻ 106 cells) were suspended in phosphatebuffered saline (PBS)–1% bovine saline solution and incubated with a
mouse antirat secondary antibody conjugated with flourescein isothiocyanate (FITC) (Pharmingen) for 30 minutes on ice (1 ␮g/106 cells). In
addition, mAb Ly-6G against the neutrophilic surface antigen GR-1
conjugated with phycoerythrin (Pharmingen) was added to gate for
neutrophilic granulocytes. PSGL-1 expression was determined on 10 000
leukocytes per mouse by means of a 4-decade FACScan with Cell Quest
software package (Becton Dickinson, San Jose, CA).
Western blotting
Bone marrow cells were flushed from the femurs and tibias of control and
core 2⫺/⫺ mice by means of cold RPMI 1640 (Gibco BRL, Grand Island,
NY) and immediately centrifuged at 1000 rpm for 5 minutes. The pellet was
washed once with cold PBS and extracted with lysis buffer (108 cells/mL)
containing 1% Triton X-100 (Fisher Scientific, Pittsburgh, PA) along with 1
mM phenylmethyl sulfonyl fluoride, 1 mM egtazic acid, and 1% protease
C2GlcNAcT-I REQUIRED FOR LEUKOCYTE ROLLING
3813
inhibitor cocktail (all Sigma Chemical, St Louis, MO) in PBS, pH 7.4.
Extraction was carried out at 4°C for 30 minutes on an orbital shaker. The
samples were clarified by centrifugation at 14 000g for 30 minutes at 4°C,
and the supernatant was transferred to fresh tubes. Samples were boiled for
5 minutes in sodium dodecyl sulfate–polymerase chain reaction sample
buffer with ␤-mercaptoethanol, electrophoresed on a 10% polyacrylamide
gel, and transferred to nitrocellulose. After blocking with 5% nonfat milk in
PBS for 1 hour at room temperature, the membrane was probed with
PSGL-1 mAb 4RA10 or rat isotype control (5 ␮g/mL, BD Pharmingen) in
2.5% nonfat milk and 0.05% Tween-20 (Fisher Scientific) in PBS for 1 hour
at room temperature, washed 3 times with PBS-Tween 20 (0.05%), and then
incubated with goat antirat IgG conjugated to horseradish peroxidase
(Pierce, Rockford, IL) for 1 hour at room temperature. After 3 washes in
PBS–Tween 20 (0.05%) followed by 1 wash in PBS, the blots were developed by
enzyme chemiluminescence by means of the substrate kit (Pierce) and were
exposed to X-omat AR5 film (Eastman Kodak, Rochester, NY).
Intravital microscopy
Mice were anesthetized with an intraperitoneal injection of ketamine (125
mg/kg body weight) (Ketalar) (Parke-Davis, Morris Plains, NJ); xylazine
(12.5 mg/kg body weight) (Phoenix Scientific, St Joseph, MO); and atropin
sulfate (0.025 mg/kg body weight) (Elkins-Sinn, Cherry Hill, NJ) and
placed on a heating pad to maintain body temperature. Some mice were
pretreated with an intrascrotal injection of recombinant murine TNF␣ as
described.6
Microscopic observations were made on an intravital microscope
(Axioskop) (Zeiss, Thornwood, NY) with a saline immersion objective
(SW 40/0.75 numerical aperture). The trachea was intubated, and one
jugular vein was cannulated for administration of anesthetic throughout the
intravital microscopic experiment. One carotid artery was cannulated for
blood pressure monitoring, blood samples, and systemic mAb injections.
Blood pressure was monitored intermittently during the experiment (model
BPMT-2) (Stemtech, Menomonee Falls, WI). During the experiment, mice
received 0.2 mL/h diluted pentobarbital in saline to maintain anesthesia and
a neutral fluid balance.
Cremaster muscle preparation
The cremaster muscle was prepared for intravital microscopy as described.6
The epididymis and testis were gently pinned to the side, exposing the
well-perfused cremaster microcirculation. The cremaster muscle was
superfused with thermocontrolled (35°C) bicarbonate-buffered saline. To
detect changes in systemic white blood cell count after injection of the
various antibodies, systemic blood samples (10 ␮L) were taken after each
mAb injection. Blood samples were diluted 1:10 with Kimura (11 mL of 5%
[wt/wt] toluidine blue. 0.8 mL of 0.03% light green SF yellowish, 0.5 mL
saturated saponin, and 5 mL of 0.07 M phosphate buffer, pH 6.4) (all from
Sigma) and were analyzed for leukocyte concentration (expressed as
number of leukocytes per microliter of whole blood). Differential leukocyte
counts were taken from blood smears by means of the Hema 3 Kit
(Biochemical Sciences, Swedesboro, NJ). To differentiate intravascular and
interstitial leukocytes into neutrophils, eosinophils, and mononuclear cells,
whole mounts of cremaster muscle were prepared as described elsewhere.25
Both rolling and firmly adhered leukocytes are visible as intravascular cells
in whole-mount histological preparations.26
Data analysis
Microvessel diameters, lengths, and rolling leukocyte velocities were
measured by means of a digital image processing system.27 Each rolling
leukocyte passing a line perpendicular to the vessel axis was counted, and
leukocyte rolling flux was expressed as leukocytes per minute. Rolling flux
fraction was calculated as described7 by dividing leukocyte rolling flux by
total leukocyte flux estimated as [WBC] ⫻ vb ⫻ ␲ ⫻ (d/2)2 where [WBC]
is the systemic leukocyte count, vb is the blood flow velocity, and d is the
venular diameter. Leukocyte rolling velocities were measured as averages
over a 2-second time window. Rolling velocities of 5 leukocytes were
measured in each venule. Centerline red blood cell velocity in the cremaster
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SPERANDIO et al
Table 1. Systemic leukocyte counts and differentials for trauma- and tumor necrosis factor-␣–induced inflammation
Systemic white
blood cell count
(cells/␮L)
Mouse
genotype
PMN cells
(%)
Lymph cells
(%)
Mononuclear
cells (%)
Eosinophils
(%)
Untreated
Core 2⫺/⫺
5200 ⫾ 800*
46 ⫾ 3*
46 ⫾ 2*
4⫾1
1 ⫾ 0.3
Control
3000 ⫾ 360*
27 ⫾ 4*
69 ⫾ 3*
2⫾1
1 ⫾ 0.4
TNF-treated
Core 2⫺/⫺
5000 ⫾ 150*
47 ⫾ 4
45 ⫾ 4
4⫾2
2⫾1
Control
2700 ⫾ 290*
48 ⫾ 8
49 ⫾ 8
2 ⫾ 0.4
1 ⫾ 0.3
All values are presented as mean ⫾ SEM.
PMN indicates polymorphonuclear; TNF, tumor necrosis factor.
*Indicates significant differences (P ⬍ .05) between core 2⫺/⫺ and control mice.
muscle preparation was measured by means of a dual photodiode and a
digital on-line cross-correlation program (Circusoft Instrumentation, Hockessin, DE). Centerline velocities were converted to mean blood flow
velocities by multiplying with an empirical factor of 0.625.28 Wall shear
rates (␥w) were estimated as 2.12 (8 vb/d), where vb is the mean blood flow
velocity, d is the diameter of the vessel, and 2.12 is a median empirical
correction factor obtained from velocity profiles measured in microvessels
in vivo.29
To specifically address the shear rate dependence of leukocyte rolling
through E-selectin, rolling flux fractions were stratified and grouped by wall
shear rate and used to construct Figure 5.
Statistics
Statistical analysis was performed with the Sigma-Stat 2.0 software
package (SPSS Science, Chicago, IL). Average vessel diameter, leukocyte
rolling flux fractions, leukocyte rolling velocities, and shear rates between
groups and treatments were compared with the one-way analysis of
variance on ranks (Kruskal-Wallis) and with a multiple pairwise comparison test (Dunn test). Leukocyte counts and differentials were compared
with the Student t test or by the Wilcoxon rank-sum test as appropriate.
Statistical significance was set at P ⬍ .05.
Results
All mice used in this work appeared healthy, active, and of normal
size and weight for their ages. The systemic leukocyte counts were
significantly higher in C2GlcNAcT-I–deficient mice than in wildtype mice. This was true for both the group without pretreatment
and the TNF␣-pretreated group (P ⬍ .05) (Table 1). The observed
increase in white blood cell count in core 2⫺/⫺ mice was due mainly
to the marked elevation in the number of neutrophils. Lymphocyte
counts were only slightly elevated in core 2⫺/⫺ mice compared with
control mice, and eosinophils and mononuclear cells showed no
difference. In the TNF␣ pretreatment group, the significantly elevated
systemic white blood cell count showed similar increases in neutrophils and lymphocytes for core 2⫺/⫺ mice and control mice.
Leukocyte rolling in untreated venules
Leukocyte rolling was analyzed in 62 venules of 11 core 2⫺/⫺ mice
and compared with rolling in 25 venules of 6 littermate controls.
Hemodynamic parameters for both groups are presented in Table 2
and show no significant differences in diameter, blood flow
velocity, shear rate, or systemic blood pressure. Leukocyte rolling
was assessed as leukocyte rolling flux fraction, which is defined as
the number of rolling leukocytes divided by the total number of
leukocytes passing through the same vessel.30 During the first hour
after exteriorization of the cremaster muscle, rolling flux fraction
was only 4% in C2GlcNAcT-I–deficient mice, compared with 27% in
control mice (P ⬍ .05) (Figure 1A). Leukocyte rolling during that time
was almost exclusively P-selectin dependent, because it was blocked by
the P-selectin mAb RB40.34 (Figure 1A). The lower leukocyte rolling
flux fraction in core 2⫺/⫺ mice can therefore be attributed to a severe
impairment in P-selectin–mediated rolling.
To investigate whether PSGL-1 was necessary for P-selectin–
dependent rolling in core 2⫺/⫺ mice in vivo, we treated C2GlcNAcTI–deficient mice with a PSGL-1–blocking mAb, 4RA10. Injection
of 4RA10 into core 2⫺/⫺ mice removed all rolling leukocytes
(Figure 1A). Injection of PSGL-1 mAb 4RA10 into control animals
led to a significant reduction in rolling flux fraction from 27% to
8%, which is similar to previous findings in rat mesenteric
venules.31 Rolling in control mice treated with mAb 4RA10 was
completely blocked by additional injection of the P-selectin–
blocking antibody RB40.34 (Figure 1A), confirming that rolling
under these conditions was completely P-selectin dependent.
Average rolling velocity (Vavg) in core 2⫺/⫺ mice was 81 ⫾ 4
␮m/s (Figure 1B) and almost 2-fold higher than in control mice
(Vavg ⫽ 44 ⫾ 3 ␮m/s) (P ⬍ .05). Control mice pretreated with
mAb 4RA10 showed an increase in rolling velocity with Vavg of
110 ⫾ 13 ␮m/s (Figure 1B), similar to the rolling velocity seen in
core 2⫺/⫺ mice. The increased rolling velocity at reduced flux
suggests that the PSGL-1–P-selectin interaction may have increased off-rates when PSGL-1 is not decorated by core 2.
Table 2. Hemodynamic and microvascular parameters in trauma- and tumor necrosis factor-␣–induced inflammation
Mouse
genotype
Mice
(n)
Venules
(n)
Diameter
(␮m)
Centerline velocity
(␮m/s)
Wall shear
rate (s⫺1)
Systemic blood pressure
(mmHg)
Untreated
Core 2⫺/⫺
Control
11
62
35 ⫾ 1
3600 ⫾ 300
1200 ⫾ 100
95 ⫾ 6
6
25
32 ⫾ 1
4000 ⫾ 400
1500 ⫾ 150
100 ⫾ 4
TNF-treated
Core 2⫺/⫺
11
55
37 ⫾ 1
2900 ⫾ 310
830 ⫾ 100
81 ⫾ 4
Control
11
43
37 ⫾ 1
3500 ⫾ 410
1020 ⫾ 120
87 ⫾ 7
Diameters, centerline velocity, wall shear rate, and systolic blood pressure are presented as mean ⫾ SEM of all investigated venules.
TNF indicates tumor necrosis factor.
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
C2GlcNAcT-I REQUIRED FOR LEUKOCYTE ROLLING
3815
Figure 2. FACS analysis of PSGL-1 on neutrophils. Control mice were treated with
increasing doses of PSGL-1–blocking mAb 4RA10 (1 ␮g, 3 ␮g, 10 ␮g, and 100 ␮g)
for more than 20 minutes. Peripheral blood was collected and processed for FACS
analysis of GR-1⫹ cells.
Short-term (2-hour) TNF␣-induced inflammation
Figure 1. Leukocyte rolling flux fraction and rolling velocities. (A) Leukocyte
rolling flux fraction in untreated cremaster venules. Effect of function-blocking mAbs
on leukocyte rolling flux fraction (mean ⫾ SEM) in venules of the cremaster muscle of
C2GlcNAcT-I–deficient mice (f) and control mice (u). * indicates significant differences (P ⬍ .05) in leukocyte rolling flux fraction between core 2⫺/⫺ and control mice.
(B) Leukocyte rolling velocities in venules of untreated core 2⫺/⫺ and control mice.
Data represent cumulative histograms of leukocyte rolling velocities measured during
the first hour after exteriorization of the cremaster muscle. Velocity distribution for
untreated core 2⫺/⫺ mice (solid line, 113 cells), untreated control mice (dotted line, 44
cells), and control mice treated with PSGL-1–blocking mAb 4RA10 (dashed line, 114
cells). Core2⫺/⫺ mice treated with 4RA10 showed no rolling.
To show that the PSGL-1–blocking mAb 4RA10 (30 ␮g) was
injected at a saturating dose, 4 control mice were treated with
increasing doses (1 ␮g, 3 ␮g, 10 ␮g, and 100 ␮g) of the
PSGL-1–blocking mAb 4RA10. Fluorescence-activated cell sorter
(FACS) analysis of blood obtained 20 minutes after injection
showed that 10 ␮g per mouse saturated all binding sites on
neutrophils, and no further increase was seen at 100 ␮g 4RA10
(Figure 2). In addition, rolling flux fraction was assessed before and
after injection of 30 ␮g 4RA10 followed by additional injection of
70 ␮g 4RA10, for a total dose of 100 ␮g 4RA10. The additional
injection led to no further drop in rolling flux fraction. Subsequent
injection of mAb RB40.34 abolished rolling completely. This
confirms that the dose of 30 ␮g 4RA10 per mouse was also
functionally saturating.
To verify that PSGL-1 was indeed significantly modified by the
C2GlcNAcT-I enzyme, we probed bone marrow neutrophils and
their precursors for PSGL-1 protein. We found an increased
mobility of PSGL-1 from core 2⫺/⫺ mice compared with littermate
control mice (Figure 3). The calculated molecular weights were
117 kd and 220 kD for the control PSGL-1 monomer and dimer and
98 kd and 188 kd for the core 2⫺/⫺ PSGL-1 monomer and dimer,
respectively.
Treatment with TNF␣ for 2 hours induces the expression of
E-selectin and enhances the expression of P-selectin on venules of
the cremaster muscle.21 We assessed leukocyte rolling in 55
venules of 11 TNF␣-treated mice lacking C2GlcNAcT-I and
compared the results with rolling in 43 venules of 11 control
animals. Hemodynamic parameters for both groups are presented
in Table 2 and show similar vessel diameters, centerline velocities,
wall shear rates, and systemic blood pressures. Leukocyte rolling
flux fraction after TNF␣ treatment was reduced to 8% in core 2⫺/⫺
mice compared with 21% in control mice (P ⬍ .05) (Figure 4),
suggesting a severe defect in selectin ligand function in C2GlcNAcTI–deficient mice.
To investigate E-selectin–mediated rolling in core 2⫺/⫺ mice,
we injected the P-selectin–blocking antibody RB40.34 into
C2GlcNAcT-I–deficient mice, which led to a decrease in rolling
flux fraction from 8% to 3% (P ⬍ .05). Treating control animals
with mAb RB40.34 led to a significant decrease in rolling flux
fraction, from 21% to 10%, which is similar to earlier findings
in P-selectin–deficient mice,6 and significantly higher than in
Figure 3. Western blot for PSGL-1. Bone marrow cells from control mice express
PSGL-1 at 117 kd and 220 kd (lane 2); core 2⫺/⫺ mice express PSGL-1 at 98 kd and
188 kd (lane 1) for the monomeric and dimeric form, respectively. PSGL-1 from core
2⫺/⫺ neutrophils shows faster mobility compared with the control bands, consistent
with absence of core 2 decoration on PSGL-1.
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SPERANDIO et al
RB40.34-treated core 2⫺/⫺ mice (P ⬍ .05). L-selectin–blocking
mAb MEL-14 had no additional effect on leukocyte rolling fluxes
in core 2⫺/⫺ and control mice (Figure 4). Adding the E-selectin–
blocking mAb 9A9 to mice treated with P-selectin–blocking mAb
RB40.34 and L-selectin–blocking mAb MEL-14 completely abolished leukocyte rolling in both core 2⫺/⫺ mice and control mice
(Figure 4). This confirms that the residual rolling seen in core 2⫺/⫺
mice and control mice treated with P-selectin–blocking mAb
RB40.34 and L-selectin–blocking mAb MEL-14 is completely
E-selectin dependent. In addition, it shows a significant impairment of E-selectin–mediated rolling in core 2⫺/⫺ mice compared
with controls.
Since the defect for E-selectin–mediated rolling in core 2⫺/⫺
mice was not complete, we investigated whether rolling was
differentially affected in venules with different wall shear rates.
Indeed, rolling was almost not affected in venules of core 2⫺/⫺ mice
treated with blocking mAbs to L- and P-selectin at wall shear rates
below 300 s⫺1, but was significantly reduced at wall shear rates
between 300 and 1000 s⫺1 (Figure 5). At very high shear rates
(greater than 1000 s⫺1), rolling flux fraction also decreased in
littermate controls (Figure 5). This analysis shows that E-selectin–
dependent rolling is not significantly affected in core 2⫺/⫺ mice at
low shear rates, which is consistent with the in vitro analysis
presented by Snapp et al (page 3806).32
To study P-selectin–dependent rolling in core 2⫺/⫺ mice, we
injected the E-selectin–blocking mAb 9A9 into core 2⫺/⫺ mice,
which resulted in a decrease in rolling flux fraction to 3% (Figure
4). This is in sharp contrast to control animals, where we observed a
dramatic increase in rolling flux fraction to 58% after injection of
9A9 (P ⬍ .05) (Figure 4), consistent with previous results.6
Next, we investigated leukocyte rolling velocities and velocity
distributions in TNF␣-treated cremaster muscle venules. TNF␣treated core 2⫺/⫺ mice showed a rolling velocity distribution
(Vavg ⫽ 11.5 ⫾ 0.6 ␮m/s) similar to the distribution in control mice
(Vavg ⫽ 10.7 ⫾ 0.8 ␮m/s) (Figure 6A). Injection of RB40.34 led to
a comparable decrease in rolling velocity in both C2GlcNAcT-I–
deficient (Vavg ⫽ 5.8 ⫾ 0.4 ␮m/s) and control mice (Vavg ⫽ 5.5 ⫾ 0.4
␮m/s) (distributions not shown). Similarly, injection of both RB40.34
and MEL14 resulted in a comparable decrease in rolling velocity in
both C2GlcNAcT-I–deficient (Vavg ⫽ 3.5 ⫾ 0.1 ␮m/s) and control
mice (Vavg ⫽ 3.8 ⫾ 0.2 ␮m/s) (Figure 6B), suggesting that Eselectin–mediated slow rolling still operates in C2GlcNAcT-I–
deficient mice. In contrast, we found a different velocity distribu-
Figure 5. E-selectin–mediated rolling in venules with different wall shear rates.
Rolling flux fraction (mean ⫾ SEM) in cremaster muscle venules of TNF␣-pretreated
core 2⫺/⫺ mice (f) and littermate control mice (u) treated with blocking mAbs against
P- and L-selectin. Data for low (less than 300 s⫺1), normal (300 to 1000 s⫺1), and high
(greater than 1000 s⫺1) wall shear rates. Results from 24 control venules and 43 core
2⫺/⫺ venules. * indicates significant differences (P ⬍ .05) between core 2⫺/⫺ mice
and control mice.
tion with a dramatic increase in rolling velocities in core 2⫺/⫺ mice
after injection of the E-selectin–blocking antibody 9A9 (Vavg ⫽
59 ⫾ 4 ␮m/s) and only a mild increase in rolling velocity in control
mice (Vavg ⫽ 14 ⫾ 1 ␮m/s) (P ⬍ .05) (Figure 6C). Leukocyte
rolling velocities after injecting mAb 9A9 into control mice were
similar to results from previous studies6 and reflect largely
P-selectin–mediated rolling. The much higher rolling velocities
found in core 2⫺/⫺ mice point toward impairment in P-selectin–
mediated leukocyte rolling.
Leukocyte differentials in cremaster muscle venules
To determine the effect of eliminating C2GlcNAcT-I on the
composition of cells in venules (intravascular) and recruited into
cremaster tissue (perivascular), we used Giemsa-stained wholemount mouse cremaster muscles to differentiate leukocytes in
TNF␣-treated mice. The number of intravascular neutrophils was
significantly reduced by about 90% in core 2⫺/⫺ mice compared
with control mice (Figure 7A). By contrast, adhesion of eosinophils
or mononuclear cells appeared to be unaffected by the absence of
C2GlcNAcT-I. To directly address the contribution of E-selectin to
leukocyte accumulation in venules, we investigated the number of
intravascular leukocytes in cremaster muscle of mice treated with
mAbs to L- and P-selectin shortly before injection of TNF␣. This
treatment did not alter leukocyte adhesion, suggesting that the
absence of C2GlcNAcT-I causes a severe reduction in neutrophil
adhesion that cannot be achieved by blocking P- and L-selectin.
Similar to the findings on intravascular leukocytes, absence of
C2GlcNAcT-I also reduced the number of extravascular neutrophils, but not eosinophils or mononuclear cells (Figure 7B).
Discussion
Figure 4. Leukocyte rolling flux fraction (mean ⴞ SEM) in TNF␣-treated
venules. Effect of function-blocking mAbs on leukocyte rolling flux fraction of
C2GlcNAcT-I–deficient mice (f) and control mice (u) 2 hours after TNF␣ injection. *
indicates significant differences (P ⬍ .05) in leukocyte rolling flux fraction between
core 2⫺/⫺ and control mice.
In this study, we have used the mouse cremaster muscle to
investigate the role of C2GlcNAcT-I in the biosynthesis and
physiological function of selectin ligands in vivo. We found that in
cremaster muscle venules of C2GlcNAcT-I–deficient mice, P- and
E-selectin–dependent rolling is sharply reduced. In addition, we
show that the observed defect in E-selectin–mediated rolling
becomes evident only at wall shear rates above 300 s⫺1.
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
C2GlcNAcT-I REQUIRED FOR LEUKOCYTE ROLLING
3817
with PSGL-1 and fucosyltransferase IV required C2GlcNAcT-I to
avidly bind fluid-phase P-selectin. Kumar et al14 found some
residual binding of soluble P-selectin to PSGL-1 when cotransfected with fucosyltransferase III but without C2GlcNAcT-I into
CHO cells. Binding was much improved by adding C2GlcNAcT-I.
Ramachandran et al33 showed that CHO cells transfected with
fucosyltransferase VII and PSGL-1 did not support rolling of a
pre-B cell line expressing P-selectin in a flow chamber system.
However, rolling was found after cotransfection with C2GlcNAcT-I.
This flow chamber assay is reversed in orientation compared with
the in vivo situation (P-selectin on the rolling cell, PSGL-1 on the
substrate), which could be of significance, since PSGL-1 is known
to be preferentially localized to the tips of microvilli on leukocytes.34 Taken together, our findings confirm and extend previous in
vitro findings to physiologically relevant wall shear rates. Interestingly, the velocity of rolling leukocytes after blocking PSGL-1 was
increased to a degree similar to that in C2GlcNAcT-I–deficient
mice. This shows that PSGL-1 in the absence of C2GlcNAcT-I
supports rolling only at a higher velocity, suggesting an increased
off-rate of the P-selectin–PSGL-1 bond in core 2⫺/⫺ mice.
The present data also show that other ligands for P-selectin may
exist and mediate some leukocyte rolling in control mice, consistent with previous findings31 and recent findings in PSGL-1–
deficient mice.35 However, there is a quantitative difference
between the degree of rolling after blocking PSGL-1. Under
Figure 6. Leukocyte rolling velocities in TNF␣-treated mice. Cumulative velocity
distribution for core 2⫺/⫺ mice (solid line) and control mice (dotted line) with no
treatment (A), P-selectin–blocking mAb RB40.34 and L-selectin–blocking mAb
MEL14 (B), and E-selectin–blocking mAb 9A9 (C). Significant velocity differences
(P ⬍ .05) between control and core 2⫺/⫺ mice were detected when E-selectin was
blocked (C). Rolling flux fraction for controls was 21%, 13%, and 58% for panels A, B,
and C, respectively, and for core 2⫺/⫺ mice, 8%, 2%, and 3%.
In untreated cremaster muscle venules of C2GlcNAcT-I–
deficient but not control mice, P-selectin–mediated rolling is
completely blocked by a mAb to PSGL-1. This is a novel finding,
demonstrating that C2GlcNAcT-I is required for optimal PSGL-1
function, but that PSGL-1 lacking core 2 oligosaccharides can still
bind to P-selectin at a rate and affinity that allow some leukocyte
rolling. The significant size difference in PSGL-1 monomer and
dimer expressed on leukocytes of core 2⫺/⫺ and control mice,
which has not been described before, is consistent with a loss of
core 2-containing glycans, suggesting that the targeted enzyme is
indeed responsible for all or most of the core 2 decoration of
PSGL-1. Consistent with our findings, flow cytometry showed that
binding of P-selectin IgM to neutrophils from core 2⫺/⫺ mice was
reduced and that neutrophils from these mice bound poorly to
immobilized P-selectin, but that binding activity was not completely abolished.16 In the accompanying report by Snapp et al,32
who used a rolling assay in a parallel-plate flow chamber, the
number of rolling neutrophils from core 2⫺/⫺ mice was reduced to
about 10% of control.
Previous in vitro work had suggested that PSGL-1 requires the
core 2 modification to bind to P-selectin.15 CHO cells transfected
Figure 7. Adherent and rolling leukocytes per square millimeter of venular
surface area and perivascular space. (A) Number of adherent and rolling
leukocytes per square millimeter of venular surface area (mean ⫾ SEM). Data are
presented for control mice (u), control mice treated with mAbs against P- (anti-P) and
L-selectin (anti-L) (䡺) and core 2⫺/⫺ mice (f). * indicates significant differences
(P ⬍ .05). (B) Number of adherent and rolling leukocytes per square millimeter of
perivascular space (mean ⫾ SEM). Data are presented for control mice (u) and core
2⫺/⫺ mice (f). * indicates significant differences from control (P ⬍ .05).
3818
BLOOD, 15 JUNE 2001 䡠 VOLUME 97, NUMBER 12
SPERANDIO et al
conditions where a mAb to P-selectin blocked almost all leukocyte
rolling, a mAb to PSGL-1 blocked only 73% in control mice,
although P-selectin–dependent rolling was reduced by 95% in
PSGL-1–deficient mice.35 Therefore, we cannot formally exclude
the possibility that mAb 4RA10, even at saturating concentrations,
may not completely block PSGL-1 function in wild-type mice.
Since PSGL-1–blocking mAb 4RA10 completely blocked rolling
in core 2⫺/⫺ mice, our findings suggest that P-selectin uses PSGL-1
and other ligands of unknown molecular identity that require
C2GlcNAcT-I.
Requirements for E-selectin–dependent rolling in vivo are not
well understood. Therefore, we addressed whether E-selectin
ligands require modification by C2GlcNAcT-I. It is clear that
E-selectin ligands must be modified by fucosyltransferase VII or IV
or another fucosyltransferase in order to be functional.36 In fact,
transfection of fucosyltransferase VII into all cell lines tested
conferred the ability to bind to E-selectin in a flow chamber
assay,37,38 suggesting that the glycoprotein requirements for Eselectin binding may be more relaxed than for P-selectin. Although
PSGL-1 has been shown to bind to E-selectin,15,39,40 recent studies
in PSGL-1–deficient mice show that PSGL-1 is not required for
neutrophil rolling through E-selectin.35 Another candidate ligand
for E-selectin, ESL-1,41 awaits confirmation of significance in vivo.
ESL-1 is decorated with N-linked but not O-linked carbohydrates.
Therefore, ESL-1 glycosylation should not be affected in
C2GlcNAcT-I–deficient mice.
The present data show that E-selectin–dependent rolling operates in core 2⫺/⫺ mice at low shear rates, but, when compared with
control mice, the number of leukocytes rolling by binding to
E-selectin is markedly reduced in venules with shear rates above
300 s⫺1. This translates into a significant recruitment defect for
neutrophils in core 2⫺/⫺ mice. Interestingly, this defect could not be
recapitulated by blocking L- and P-selectin in control mice (Figure
7A), suggesting that C2GlcNAcT-I activity is indeed necessary for
a functionally important E-selectin ligand on neutrophils. It is
possible that other, C2GlcNAcT-I–independent E-selectin ligands
exist on eosinophils and mononuclear cells. However, the present
data do not unequivocally show this, because eosinophils and
mononuclear cells also use selectin-independent pathways for
rolling and adhesion.42 Our result are in agreement with previous
findings of reduced core 2⫺/⫺ neutrophil binding to E-selectin IgG
chimeras in a flow chamber detachment assay and reduced binding
of neutrophils to E-selectin IgM chimeras in a flow cytometric
assay.16 However, the accompanying report did not find an
impairment of E-selectin ligand function in a parallel-plate flow
chamber attachment and rolling assay coated with E-selectin
transfectants.32 In this assay, low shear stress (1.5 dyne/cm2) was
used to allow for neutrophil attachment under in vitro conditions.
In vitro, neutrophil attachment is reduced because of geometric
differences to in vivo conditions43 and the absence of red blood
cells.44 Under in vivo conditions, we observed shear rates in the
range of 150 to 1500 s⫺1, which approximately correspond to wall
shear stress levels of 1.5 to 15 dyne/cm2. In agreement with the
accompanying results from the in vitro assay,32 we found very little
decrease in E-selectin–dependent rolling at wall shear rates below
300 s⫺1, when the rolling flux fraction almost reached control
values. This finding suggests an explanation for the difference in
E-selectin–dependent rolling between the accompanying report32
and the flow chamber detachment assay presented by Ellies et al.16
We conclude that E-selectin–dependent rolling under low shear
conditions is similar in the presence or absence of C2GlcNAcT-I,
whereas E-selectin–dependent rolling is impaired in the absence
of C2GlcNAcT-I at normal shear rates, resulting in reduced
neutrophil accumulation in TNF␣-treated venules. Interestingly,
the ratio of perivascular neutrophils to intravascular neutrophils
is very similar for both core 2⫺/⫺ mice and control mice. This
suggests that the rate of transmigration is not affected by the
absence of C2GlcNAcT-I. Rather, the defect in neutrophil
migration can most likely be attributed to the dramatic decrease
in neutrophil rolling flux.
In conclusion, the present study has identified some of the
mechanisms by which the absence of C2GlcNAcT-I impairs
neutrophil recruitment to sites of inflammation.16 We conclusively
show (1) that P-selectin–dependent rolling via PSGL-1 is severely
impaired, (2) that rolling through other P-selectin ligands is
completely absent in core 2⫺/⫺ mice, and (3) that, on the basis of
direct evidence, E-selectin–dependent rolling under physiological
flow conditions is impaired in core 2⫺/⫺ mice at higher wall shear
rates. It is possible that other defects exist in core 2⫺/⫺ mice that
might interfere with normal signaling, adhesion, or transmigration
of neutrophils. However, the severe functional deficits in leukocyte
rolling appear to be sufficient to explain the severe decrease of
neutrophil recruitment found in core 2⫺/⫺ mice.
Acknowledgments
We thank Drs Ruth Eytner, Martin Wild, and Dietmar Vestweber,
Universität Münster, Germany, for providing mAbs RB40.34 and
4RA10, and Dr Barry Wolitzky for providing mAb 9A9.
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